Agents that are effective in killing neoplastic and other diseased or abnormal cells generally cannot be administered to a patient in therapeutically effective doses, because they also exert cytotoxic effects on normal cells. Therapeutic protocols for treating cancer and other disorders using cytotoxic agents such as toxins, drugs, radioisotopes and the like, are generally limited by the toxicity of the cytotoxic agent to normal cells and tissues. Efforts have therefore been directed to linking cytotoxic therapeutic agents to carriers, such as antibodies, which have an affinity and specificity for certain target tissues, cells and antigens.
Recent research efforts in the field of tumor immunology have identified antibodies to antigenic determinants expressed preferentially on tumor cells. Such antibodies may be employed as carriers for cytotoxic agents to provide selective delivery of cytotoxic agents to target tissues. Antibodies, fragments thereof and the like have also been utilized as carriers for diagnostic agents such as diagnostic radioisotopes, to provide highly selective delivery of the diagnostic agent to the target tissue, thereby providing enhanced imaging properties. In vivo administration of diagnostic and therapeutic immunoconjugates comprising an effector moiety exhibiting diagnostic or therapeutic properties and a carrier moiety exhibiting specificity and affinity for target tissues, cells, antigens, or the like are believed to be of tremendous potential in diagnosis and treatment of cancer and a variety of other diseases.
Development of techniques for generating monoclonal antibodies having specificity for a single epitope has further expanded the potential for immunoconjugates as in vivo diagnostic and therapeutic agents. One of the problems associated with in vivo administration of immunoconjugates to patients for diagnostic or therapeutic purposes is that the immunoconjugate itself, or some portion thereof, may stimulate a humoral immune response in the patient. This problem may arise frequently, since antibodies raised in non-human species are typically employed in diagnostic and therapeutic immunoconjugates for in vivo administration in humans. Stimulation of a humoral immune response will result in production of serum antiglobulin and may result in formation of immune complexes comprising serum antiglobulin and the immunoconjugate administered. Formation of immune complexes may seriously hamper the efficacy of the product, and may, in some cases, pose a serious health hazard to the patient.
Formation of immune complexes comprising the immunoconjugate, or a portion thereof, bound to circulating antiglobulin as a result of in vivo administration of diagnostic or therapeutic immunoconjugates may affect the biodistribution and clearance rate of the immunoconjugates. In general, formation of immune complexes reduces the amount of immunoconjugate available for diagnostic or therapeutic purposes and results in retention of the administered immunoconjugate in non-target tissues. With respect to diagnostic immunoconjugates, stimulation of a humoral immune response and formation of circulating immune complexes is detrimental primarily because it disperses the diagnostic agent, and thereby reduces imaging clarity. For therapeutic immunoconjugates, however, the detrimental effects of in vivo immunoconjugate administration may be considerably more serious, since formation of circulating immune complexes comprising cytotoxic agents not only reduces the cytotoxic effect of the immunoconjugate on the target cell population, but it localizes cytotoxic agents in non-target tissues such as the liver and kidneys, and these tissues may be seriously damaged.
Where the carrier moiety of the immunoconjugate is derived from a species different from that of the patient, the probability of stimulating antiglobulin production in the patient is very high. In efforts to reduce the immunogenicity of immunoconjugates comprising carrier moieties derived from non-human sources, antibody fragments have been used to quantitatively reduce the total amount of foreign protein, while providing the same or increased levels of binding capacity. Use of antibody fragments such as Fab, Fab', F(ab').sub.2, and the like is well known in the art. In addition, chimeric antibodies have been developed in efforts to reduce the immunogenicity of carrier moieties derived from non-human sources. Chimeric antibodies may comprise specificity determining regions derived from non-human sources, while other portions of the antibody, such as the constant regions, are of human origin. Immunogenicity of conjugates comprising non-human antibody constituents, however, remains a serious problem and limits the diagnostic and therapeutic application of immunoconjugates. Anti-allotype reactivity to the human constant domains of chimeric antibodies may also affect the efficacy of immunoconjugates comprising chimeric carrier moieties
Conventional immunoassays for detecting antibodies and antigens include enzyme immunoassays such as the ELISA (enzyme-linked immunosorbent assay) protocol, radioimmunoassays such as the RIA-immunoprecipitation assay, and immunofluorescence protocols. Typically, a predetermined quantity of antigen (or antibody) is adsorbed on a solid phase, protein binding surface. The test sample to be assayed for antibodies (antigens) is then contacted to the surface having antigen (antibody) bound thereto, and antibodies (antigens) in the test sample bind to the immobilized antigen (antibody). Radioactive or enzyme-labeled immunoglobulin probes are then contacted to the surface and bind to the immobilized antibodies (antigens). The amount of labeled probe bound to the solid support can be quantitated and is indicative of the antibody (antigen) concentration in the test sample.
ELISA protocols typically involve multiple microassays utilizing several dilutions of human serum and a single target antigen (antibody) concentration. Microtiter plates are typically used for performing the multiple microassays necessary to detect the presence of antibody (antigen). ELISA protocols require extensive handling and manipulation of samples and reagents, which may substantially reduce the accuracy of the assay results. In fact, coefficients of variance of up to 25% and 30% are not unusual for ELISA results. This level of accuracy may be unacceptable for many applications, and particularly for applications involving evaluation of patients for therapeutic protocols utilizing immunoconjugates comprising radionuclides, toxins and the like. In addition, spectrophotometric equipment such as a microplate reader is effectively required for accurate analysis of the assay results, since manual analysis significantly reduces accuracy of the assay results. Such equipment requires a substantial capital investment, which may not be practicable unless assays are performed on a relatively large, commercial scale.
Disadvantages of using radioimmunoassay procedures include the necessity of extensive sample manipulations, including multiple dilutions, incubations and washing steps. In addition, a potentially hazardous radioisotope such as .sup.125 I is employed. Processing samples according a radioimmunoassay protocol consumes at least several hours, and requires relatively complex laboratory equipment and skilled technicians. Immunofluorescent staining generally provides an accurate indication of specificity, and it permits visualization of the antigen-antibody reaction. Immunofluorescence methodologies, however, are time consuming and difficult to perform on a large scale. Moreover, analysis of immunofluorescence assay results requires the analytical judgment of experienced technicians.